Screening the secondary metabolome of an unidentified basidiomycete for antifungal compounds

Daniel Schwenk1, Uwe Horn2, Dirk Hoffmeister1,

1Friedrich-Schiller-Universitt

Jena, Department of Pharmaceutical Biology at the Hans-Knll-Institute, Beutenbergstrae 11a, 07745 Jena, dirk.hoffmeister@hki-jena.de 2Bio Pilot Plant, Hans-Knll-Institute, Beutenbergstrae 11a, 07745 Jena

Background
An unusual basidiomycete ("BY") isolated from wood has so far defied any attempts to identify the genus, based on both morphological and genetic features. Sequencing of ITS regions points to the order Russulales. As the fungus exerts strong antifungal effects, further research was warranted. A series of natural products has been isolated, structurally elucidated and tested for antifungal activity. Preliminary results suggest that the bioactivity of "BY" is not an effect of a single agent, but due to several structurally dissimilar compounds.
Figure 1: "BY" on Malt-Peptone-Agar

Results
While separation of the bioactive crude extract by retention time on RPHPLC yielded only inactive samples (Fig. 3A), the recombination of these samples restores the bioactivity (Fig. 3B).
separation A)

B)

recombination

Figure 2: RP-HPLC-UV chromatogram of crude extract, =254 nm

Figure 3: A) Lost bioactivity against A.nidulans after fractionation via RPHPLC and B) reconstituted fractions of previously inactive samples. Numbers indicate omitted fractions from Fig. 3A.

Various natural products have been isolated, chemically characterized and evaluated for their antifungal properties. Our results indicate that the bioactive secondary metabolites of "BY" fall into two structurally dissimilar families of compounds: i) vibralactone1) and derivatives (Fig. 5), and ii) fomannoxin2) and derivatives (Fig. 7). Both backbone structures have been found in other species of the russuloid clade as well (Boreostereum vibrans and Heterobasidium annosum, respectively), thus supporting our estimated phylogenetic placement of "BY". A third class of compounds, closely resembling piptoporic acid3) isolated from Piptoporus australiensis, did not contribute to the antifungal activity but is responsible for the yellow pigmentation (Fig. 1).

i) Vibralactone-related compounds

ii) Fomannoxin-related compounds

possible oxidation: hydroxy- or ketone group

Figure 6 (above): Polyolefinic pigments produced by "BY". n=5: piptoporic acid

possible double bond positions

Figure 5: The unusual fused -lactone vibralactone derivatives found in "BY" .

and

ring cleavage possible

possible hydroxylation

Figure 7 (left): By prenylation and subsequent oxidative modification of different starter molecules originating from shikimic acid pathway, metabolic diversity is created.

Perspectives
Future work will include i) investigations towards a potential synergistic activity of antifungal compounds, ii) target identification through microarray experiments and 14C-labeled precursor uptake assays with exposed A. nidulans cells to initially investigate the mode of action of the above shown natural products, iii) chemical synthesis and derivatization of antifungal structures to assign structure-activity relationships.
Acknowledgment
We gratefully acknowledge Dr. Markus Nett and Andrea Perner (HKI Jena), respectively, for recording NMR and high resolution mass spectra. This research project is funded by the International Leibniz Research School for Microbial and Biomolecular Interactions (ILRS Jena).

References
1) DZ Liu et al.: Org. Lett., 2006, 8, 5749-5752 2) DMX Donnelly et al.: J. Chem. Soc., Perkin Trans. 1, 1980, 2196-2199 3) M Gill: J. Chem. Soc., Perkin Trans. 1, 1982, 1449-1453